DESCRIPTION: (provided by the applicant) Presynaptic dopamine (DA) transporters (DATs) constitute the primary mechanism for inactivation of DA in the brain. DAT proteins are high-affinity targets for important addictive and therapeutic drugs including cocaine, amphetamines and methylphenidate (Ritalin). DATs are subject to significant regulatory modulation but molecular mechanisms supporting DAT regulation are unknown. Recent studies indicate that DATs are acutely regulated by coordinated mechanisms involving kinase activation, transporter phosphorylation and altered membrane trafficking/stabilization, though as yet genes responsible for this regulation remain to be identified. We have established a model system permitting DAT evaluation in a native neuronal context, exploiting the powerful transgenic and genomic tools afforded in Caenorhabditis elegans. In this system, we have the unique ability to selectivelv introduce or inactivate genes in living DA neurons, allowing us to test specific hypotheses regarding DAT structure/function, drug modulation and drug and kinase triggered DAT regulation. Moreover, a novel cell culture approach has been established that permits a detailed electrophysiologic and optical analysis of DAT function and regulation in identified DA neurons and that can be scaled to support the identification of novel DAT regulatory genes. In our CEBRA proposal, we seek to 1) analyze the functional and regulatory pathways supporting C. elegans DAT (CeDAT) expression in cultured DA neurons and 2) to develop a system where a combination of transgenic and proteomic approaches can be applied to permit the evaluation of hypothesized and novel DAT regulators.